In order to analyse copper-induced calcium release and (reactive oxygen species) ROS accumulation and their role in antioxidant and defense enzymes activation, the marine alga Ulva compressa was exposed to 10 mM copper for 7 d. The level of calcium, extracellular hydrogen peroxide (eHP), intracellular hydrogen peroxide (iHP) and superoxide anions (SA) as well as the activities of ascorbate peroxidase (AP), glutathione reductase (GR), glutathione-S-transferase (GST), phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) were determined. Calcium release showed a triphasic pattern with peaks at 2, 3 and 12 h. The second peak was coincident with increases in eHP and iHP and the third peak with the second increase of iHP. A delayed wave of SA occurred after day 3 and was not accompanied by calcium release. The accumulation of iHP and SA was mainly inhibited by organellar electron transport chains inhibitors (OETCI), whereas calcium release was inhibited by ryanodine. AP activation ceased almost completely after the use of OETCI. On the other hand, GR and GST activities were partially inhibited, whereas defense enzymes were not inhibited. In contrast, PAL and LOX were inhibited by ryanodine, whereas AP was not inhibited. Thus, copper stress induces calcium release and organellar ROS accumulation that determine the differential activation of antioxidant and defense enzymes.

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High-quality protein extracts are required for proteomic studies, a field that is poorly developed for marine macroalgae. A reliable phenol extraction protocol using Scytosiphon gracilis Kogame and Ectocarpus siliculosus (Dillwyn) Lyngb. (Phaeophyceae) as algal models resulted in high-quality protein extracts. The performance of the new protocol was tested against four methods available for vascular plants and a seaweed. The protocol, which includes an initial step to remove salts from the algal tissues, allowed the use of highly resolving two-dimensional gel electrophoresis (2-DE) protein analyses, providing the opportunity to unravel potentially novel physiological processes unique to this group of marine organisms.

In order to identify genes/proteins involved in copper tolerance, the marine alga Ulva compressa was cultivated with 10 μM copper for 3 days. The activities of antioxidant enzymes ascorbate peroxidase (AP), peroxiredoxin (PRX), thioredoxin (TRX), and glutathione-S-transferase (GST) and the level of lipoperoxides were determined in the alga cultivated with and without copper addition. Antioxidant enzyme activities and lipoperoxides level increased in response to copper excess, indicating that the alga was under oxidative stress. A cDNA library was prepared using U. compressa cultivated with 10 μM copper for 3 days. A total of 3 × 10(4) clones were isolated and 480 clones were sequenced, resulting in 235 non-redundant ESTs, of which 104 encode proteins with known functions. Among them, we identified proteins involved in (1) antioxidant metabolism such as AP, PRX, TRX, GST, and metalothionein (MET), (2) signal transduction, such as calmodulin (CAM), (3) calcium-dependent protein kinase (CDPK) and nucleoside diphosphate kinase (NDK), (4) gene expression, (5) protein synthesis and degradation, and (6) chloroplast and mitochondria electron transport chains. Half of the identified proteins are potentially localized in organelles. The relative level of 18 genes, including those coding for AP, PRX, TRX, GST, MET, CAM, CDPK, and NDK were determined by quantitative RT-PCR in the alga cultivated with 10 μM copper for 0 to 7 days. Transcript levels increased in response to copper stress and most of them reached a maximum at days 3 and 5. Thus, the selected genes are induced by copper stress and they are probably involved in copper acclimation and tolerance.

The taxonomic status of an alga with complanate thalli, occurring in central Chile and belonging to the genus Scytosiphon, was elucidated. Morphological and molecular features demonstrated that, in addition to the known and widespread constricted S. lomentaria (Lyngb.) Link that occurs along the Chilean coast, there is a Scytosiphon with complanate thalli that occurs only in central Chile-S. gracilis Kogame. Morphological analyses of this previously unreported complanate Scytosiphon showed thalli without constrictions, coherent plurilocular sporangia without ascocysts, and phaeophycean hairs arising from cortical cells. Furthermore, sequences of the internal transcribed spacer (ITS1 and ITS2) regions of the complanate Scytosiphon were 99.8% and 100% identical to those detected in S. gracilis from Korea. ITS-based comparative analyses showed that complanate Scytosiphon grouped in a different clade than S. lomentaria and S. tenellus Kogame from various parts of the world, including Chilean species. Moreover, molecular analyses suggest the occurrence of two distinct ITS types of S. lomentaria in northern Chile, corresponding to the Korean and Greek types. On the other hand, biochemical analyses of copper-induced antioxidant responses in S. gracilis and S. lomentaria showed an identical increase in antioxidant enzyme activities. These results suggest that copper tolerance might be a constitutive trait in these species of Scytosiphon.